共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
Tomomasa Watanabe Nobuaki Ogasawara 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,717(3):439-444
Three trypsinogens and one chymotrypsinogen were found in and purified from the pancreas of a mouse strain (CFO). The molecular weights of the trypsinogens and the chymotrypsinogen were all estimated as 25 000. The enzyme properties of the three trypsinogens were studied and they showed very low Km values (3.2?6.5 μM) for the substrates, BzArgOEt and TosArgOMe, and the dame pH optimum profile between pH 8.0–10.0. However, the ratios of catalytic rate constants, kcat (s?1), with BzArgOEt as substrates compared to that with TosArgOMe were very different. The values of Try-III were similar with the two substrates, Try-I was slightly higher value with TosArgOMe than with BzArgOEt, and the values of Try-II were much higher with TosArgOMe than with BzArgOEt. Also, the trypsinogens and the chymotrypsinogen were purified from pancreas of Mol-A strain mice. When the enzyme properties of the three trypsinogens were examined, one form of trypsinogen (Try-I) was shown to have different properties in kcat (s?1) for the two substrates, compared to the trypsinogen of CFO mice. 相似文献
3.
Masayuki Okuyama Takuya Yoshida Hironori Hondoh Haruhide Mori Min Yao Atsuo Kimura 《FEBS letters》2014
The role of calcium ion in the active site of the inverting glycoside hydrolase family 97 enzyme, BtGH97a, was investigated through structural and kinetic studies. The calcium ion was likely directly involved in the catalytic reaction. The pH dependence of kcat/Km values in the presence or absence of calcium ion indicated that the calcium ion lowered the pKa of the base catalyst. The significant decreases in kcat/Km for hydrolysis of substrates with basic leaving groups in the absence of calcium ion confirmed that the calcium ion facilitated the leaving group departure. 相似文献
4.
Takayoshi Tagami Masayuki Okuyama Hiroyuki Nakai Young-Min Kim Haruhide Mori Kazunori Taguchi Birte Svensson Atsuo Kimura 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(1):329-335
Glycoside hydrolase family 31 α-glucosidases (31AGs) show various specificities for maltooligosaccharides according to chain length. Aspergillus niger α-glucosidase (ANG) is specific for short-chain substrates with the highest kcat/Km for maltotriose, while sugar beet α-glucosidase (SBG) prefers long-chain substrates and soluble starch. Multiple sequence alignment of 31AGs indicated a high degree of diversity at the long loop (N-loop), which forms one wall of the active pocket. Mutations of Phe236 in the N-loop of SBG (F236A/S) decreased kcat/Km values for substrates longer than maltose. Providing a phenylalanine residue at a similar position in ANG (T228F) altered the kcat/Km values for maltooligosaccharides compared with wild-type ANG, i.e., the mutant enzyme showed the highest kcat/Km value for maltotetraose. Subsite affinity analysis indicated that modification of subsite affinities at + 2 and + 3 caused alterations of substrate specificity in the mutant enzymes. These results indicated that the aromatic residue in the N-loop contributes to determining the chain-length specificity of 31AGs. 相似文献
5.
Marcelo M. Pedroso Fernanda Ely Thierry Lonhienne Lawrence R. Gahan David L. Ollis Luke W. Guddat Gerhard Schenk 《Journal of biological inorganic chemistry》2014,19(3):389-398
Binuclear metallohydrolases are a large and diverse family of enzymes that are involved in numerous metabolic functions. An increasing number of members find applications as drug targets or in processes such as bioremediation. It is thus essential to have an assay available that allows the rapid and reliable determination of relevant catalytic parameters (k cat, K m, and k cat/K m). Continuous spectroscopic assays are frequently only possible by using synthetic (i.e., nonbiological) substrates that possess a suitable chromophoric marker (e.g., nitrophenol). Isothermal titration calorimetry, in contrast, affords a rapid assay independent of the chromophoric properties of the substrate—the heat associated with the hydrolytic reaction can be directly related to catalytic properties. Here, we demonstrate the efficiency of the method on several selected examples of this family of enzymes and show that, in general, the catalytic parameters obtained by isothermal titration calorimetry are in good agreement with those obtained from spectroscopic assays. 相似文献
6.
Steady-state and pre-steady-state kinetics for the hydrolysis of p-nitrophenyl esters of N-α-carbobenzoxy(-l-)amino acids catalyzed by leucine-proteinase were determined between pH 5 and 10 (I = 0.1 molar) at 23 ± 0.5°C. For the substrates considered: (a) the acylation step is rate-limiting in catalysis; (b) the pH profiles of kcat and kcat/Km reflect the ionization of two groups with pKa values ranging between 6.5 and 6.9, and 8.1 and 8.3 (probably, the histidine residue involved in the catalytic triad and the N-terminus, respectively); and (c) values of Km are pH independent. Among the substrates examined, N-α-carbobenzoxy-l-leucine-p-nitrophenyl ester shows the most favorable catalytic parameters and allows to determine an enzyme concentration as low as 5 × 10−10 molar at the optimum pH value (approximately 7.5). 相似文献
7.
Horse liver alcohol dehydrogenase SS: purification and characterization of the homogenous isoenzyme.
Alcohol dehydrogenase SS was prepared from horse liver by salt fractionation, ion-exchange chromatography, and affinity chromatography. The purified isoenzyme is free from extraneous protein and other alcohol dehydrogenase isoenzyme contaminants and contains four Zinc atoms per molecule. The substrate specificity with saturated aliphatic alcohols and aldehydes of two to six carbon chain lengths has been investigated. The Km values and turnover numbers at maximal velocity (kcat) are presented. Values of kcat are constant within a substrate category and independent of the substrate chain length, while the Km values decrease with the increase of the substrate chain length. The Km values for two- and three-carbon substrates are large, that for ethanol (40 mm) is two orders of magnitude larger than that reported for classical preparations of horse liver alcohol dehydrogenase. At pH 7, the kcat values for alcohol oxidation are almost 30 times smaller than for aldehyde reduction. The enzyme has been characterized with regard to specific activity with several nonsteroidal substrates and with two steroids: 3-oxo-5β-androstan-17β-ol and 5β-pregnan-21-ol-3,20-dione hemisuccinate. NAD(H) is the preferred coenzyme. Values of Km for NADH with constant steroidal substrates are an order of magnitude smaller than the corresponding Km values with nonsteroidal substrates. A possible explanation for this observation is presented. 相似文献
8.
《Bioorganic & medicinal chemistry letters》2019,29(17):2452-2454
Protein tyrosine phosphatase (PTP) targeted, peptide based chemical probes are valuable tools for studying this important family of enzymes, despite the inherent difficulty of developing peptides targeted towards an individual PTP. Here, we have taken a rational approach to designing a SHP-2 targeted, fluorogenic peptide substrate based on information about the potential biological substrates of SHP-2. The fluorogenic, phosphotyrosine mimetic phosphocoumaryl aminopropionic acid (pCAP) provides a facile readout for monitoring PTP activity. By optimizing the amino acids surrounding the pCAP residue, we obtained a substrate with the sequence Ac-DDPI-pCAP-DVLD-NH2 and optimized kinetic parameters (kcat = 0.059 ± 0.008 s−1, Km = 220 ± 50 µM, kcat/Km of 270 M−1s−1). In comparison, the phosphorylated coumarin moiety alone is an exceedingly poor substrate for SHP-2, with a kcat value of 0.0038 ± 0.0003 s−1, a Km value of 1100 ± 100 µM and a kcat/Km of 3 M−1s−1. Furthermore, this optimized peptide has selectivity for SHP-2 over HePTP, MEG1 and PTPµ. The data presented here demonstrate that PTP-targeted peptide substrates can be obtained by optimizing the sequence of a pCAP containing peptide. 相似文献
9.
Ismael Bustos-Jaimes Rodrigo Mora-Lugo Mario L. Calcagno Amelia Farrés 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(12):2222-2227
Lipases are useful catalysts for a wide variety of industrial purposes. Herein we report the stability and thermal dependence of the activity of wild-type Bacillus pumilus lipase (BplA) and four site-directed mutants designed to improve its thermal stability. The Gly28:Ser mutation produces a dramatic four-fold increase in its kcat and a remarkable increase in its stability. While the increase in kcat is temperature-independent, the increase in stability shows that the resultant interactions of this mutation have a strong enthalpic component. Thermal dependence of stability, kcat, KM and kcat/KM were analysed to gain insight on the structural effects of mutations on BplA. Our results are consistent with a gain in enzyme mobility for those mutants displaying enhanced catalytic properties; the analysis of thermal dependence of kinetic parameters indicates that the mutations did not change either the catalytic mechanism or the rate-limiting step of catalysis. 相似文献
10.
A series of substituted kynurenines (3-bromo-dl, 3-chloro-dl, 3-fluoro-dl, 3-methyl-dl, 5-bromo-l, 5-chloro-l, 3,5-dibromo-l and 5-bromo-3-chloro-dl) have been synthesized and tested for their substrate activity with human and Pseudomonas fluorescens kynureninase. All of the substituted kynurenines examined have substrate activity with both human as well as P. fluorescens kynureninase. For the human enzyme, 3- and 5-substituted kynurenines have kcat and kcat/Km values higher than l-kynurenine, but less than that of the physiological substrate, 3-hydroxykynurenine. However, 3,5-dibromo- and 5-bromo-3-chlorokynurenine have kcat and kcat/Km values close to that of 3-hydroxykynurenine with human kynureninase. The effects of the 3-halo substituents on the reactivity with human kynureninase may be due to electronic effects and/or halogen bonding. In contrast, for the bacterial enzyme, 3-methyl, 3-halo and 3,5-dihalokynurenines are much poorer substrates, while 3-fluoro, 5-bromo, and 5-chlorokynurenine have kcat and kcat/Km values comparable to that of its physiological substrate, l-kynurenine. Thus, 5-bromo and 5-chloro-l-kynurenine are good substrates for both human as well as bacterial enzyme, indicating that both enzymes have space for substituents in the active site near C-5. The increased activity of the 5-halokynurenines may be due to van der Waals contacts or hydrophobic effects. These results may be useful in the design of potent and/or selective inhibitors of human and bacterial kynureninase. 相似文献
11.
Type A botulinum neurotoxin is one of the most lethal of the seven serotypes and is increasingly used as a therapeutic agent
in neuromuscular dysfunctions. Its toxic function is related to zinc-endopeptidase activity of the N-terminal light chain
(LC) on synaptosome-associated protein-25 kDa (SNAP-25) of the SNARE complex. To understand the determinants of substrate
specificity and assist the development of strategies for effective inhibitors, we used site-directed mutagenesis to investigate
the effects of 13 polar residues of the LC on substrate binding and catalysis. Selection of the residues for mutation was
based on a computational analysis of the three-dimensional structure of the LC modeled with a 17-residue substrate fragment
of SNAP-25. Steady-state kinetic parameters for proteolysis of the substrate fragment were determined for a set of 16 single
mutants. Of the mutated residues non-conserved among the serotypes, replacement of Arg-230 and Asp-369 by polar or apolar
residues resulted in drastic lowering of the catalytic rate constant (k
cat), but had less effect on substrate affinity (K
m). Substitution of Arg-230 with Lys decreased the catalytic efficiency (k
cat/K
m) by 50-fold, whereas replacement by Leu yielded an inactive protein. Removal of the electrostatic charge at Asp-369 by mutation
to Asn resulted in 140-fold decrease in k
cat/K
m. Replacement of other variable residues surrounding the catalytic cleft (Glu-54, Glu-63, Asn-66, Asp-130, Asn-161, Glu-163,
Glu-170, Glu-256), had only marginal effect on decreasing the catalytic efficiency, but unexpectedly the substitution of Lys-165
with Leu resulted in fourfold increase in k
cat/K
m. For comparison purposes, two conserved residues Arg-362 and Tyr-365 were investigated with substitutions of Leu and Phe,
respectively, and their catalytic efficiency decreased 140- and 10-fold, respectively, whereas substitution of the tyrosine
ring with Asn abolished activity. The altered catalytic efficiencies of the mutants were not due to any significant changes
in secondary or tertiary structures, or in zinc content and thermal stability. We suggest that, despite the large minimal
substrate size for catalysis, only a few non-conserved residues surrounding the active site are important to render the LC
competent for catalysis or provide conformational selection of the substrate. 相似文献
12.
An acid carboxypeptidase (EC 3.4.16.1) has been isolated from the culture filtrate of a wood-degrading Basidiomycete, Pycnoporus sanguineus and the molecular and enzymatic properties of the enzyme were determined. The extracellular acid carboxypeptidase was homogeneous on polyacrylamide gel electrophoresis at pH 9.4 and SDS-disc gel electrophoresis. The MWs as determined by gel filtration and SDS-gel electrophoresis were 50 000 and 54 000, respectively. The isoelectric point was pH 4.78 using electrofocusing. The purified enzyme had a pH optimum of 3.4, a Km of 0.74 mM and a kcat of 16/sec with benzyloxycarbonyl-l-glutamyl-l-tyrosine. The Km and kcat values for bradykinin at pH 3.4 and 30° were 2.0 mM and 25/sec. Values for angiotensin at pH 3.4 and 30° were 0.76 mM and 2.4/sec, respectively. 相似文献
13.
《Bioscience, biotechnology, and biochemistry》2013,77(8):1703-1708
It is a common practice to employ k cat[E]0/K m as a first-order rate constant for the analysis of an enzymatic reaction, where [E]0 is the total enzyme concentration. I describe in this report a serious shortcoming in analyzing enzymatic reactions when k cat[E]0/K m is employed and show that k cat[E]0/K m can only be applied under very limited conditions. I consequently propose the use of a more universal first-order rate constant, k cat[ES]K/[S]0, where [ES]K is the initial equilibrium concentration of the ES-complex derived from [E]0, [S]0 and K m. Employing k cat[ES]K/[S]0 as the first-order rate constant enables all enzymatic reactions to be reasonably simulated under a wide range of conditions, and the catalytic and binding contributions to the rate constant of any enzyme can be determined under any and all conditions. 相似文献
14.
The serine alkaline protease, SAPB, from Bacillus pumilus CBS is characterized by its high thermoactivity, pH stability and high catalytic efficiency (kcat/Km) as well as its excellent stability and compatibility with an alkaline environment under harsh washing conditions. Based on sequence alignments and homology-modeling studies, the present study identified five amino acids Leu31, Thr33, Asn99, Phe159 and Gly182 being putatively important for the enzymatic behaviour of SAPB. To corroborate the role of these residues, 12 mutants were constructed by site-directed mutagenesis and then purified and characterized. The findings demonstrate that the single mutants F159T, F159S and G182S and combined double substitutions were implicated in the decrease of the optimum pH and temperature to 8.0–9.0 and 50 °C, respectively, and that mutant F159T/S clearly affected substrate affinity and catalytic efficiency. With regards to the single L31I, T33S and N99Y and combined double and triple mutations, the N99Y mutation strongly improved the half-life times at 50 °C and 60 °C to 660 and 295 min from of 220 and 80 min for the wild-type enzyme, respectively. More interestingly, this mutation also shifted the optimum temperature from 65 °C to 75 °C and caused a prominent 31-fold increase in kcat/Km with N-succinyl-l-Ala-Ala-Pro-Phe-p-nitroanilide (AAPF). The L31I and T33S mutants were observed to improve mainly the optimum pH from 11.0 to 11.5 and from 11.0 to 12.0, respectively. Kinetic studies of double and triple mutants showed that the cumulative effect of polar uncharged substitutions had a synergistic effect on the P1 position preference using synthetic peptide substrates, which confirms the implication of these amino acids in substrate recognition and catalytic efficiency. 相似文献
15.
This work addresses the binding, cleavage and dissociation rates for the substrate and products of a synthetic RNaseA mimic that was combinatorially selected using chemically modified nucleoside triphosphates. This trans-cleaving DNAzyme, 925-11t, catalyzes sequence-specific ribophosphodiester hydrolysis in the total absence of a divalent metal cation, and in low ionic strength at pH 7.5 and in the presence of EDTA. It is the first such sequence capable of multiple turnover. 925-11t consists of 31 bases, 18 of which form a catalytic domain containing 4 imidazole and 6 allylamino modified nucleotides. This sequence cleaves the 15 nt long substrate, S1, at one embedded ribocytosine at the eighth position to give a 5′-product terminating in a 2′,3′-phosphodiester and a 3′-product terminating in a 5′-OH. Under single turnover conditions at 24°C, 925-11t displays a maximum first-order rate constant, kcat, of 0.037 min−1 and a catalytic efficiency, kcat/Km, of 5.3 × 105 M−1 min−1. The measured value of kcat under catalyst excess conditions agrees with the value of kcat observed for steady-state multiple turnover, implying that slow product release is not rate limiting with respect to multiple turnover. The substrate specificity of 925-11t was gauged in terms of kcat values for substrate sequence variants. Base substitutions on the scissile ribose and at the two bases immediately downstream decrease kcat values by a factor of 4 to 250, indicating that 925-11t displays significant sequence specificity despite the lack of an apparent Watson–Crick base-pairing scheme for recognition. 相似文献
16.
Shuhuai Yu Lanjun Zhu Chen Zhou Tao An Tao Zhang Bo Jiang Wanmeng Mu 《Applied microbiology and biotechnology》2014,98(4):1621-1630
The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat/K m) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s?1, and 2.83 mM?1 s?1, respectively. The K m for NAD+ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate. 相似文献
17.
Contributions of binding and catalytic rate constants to evolutionary modifications inKm of NADH for muscle-type (M4) lactate dehydrogenases 总被引:1,自引:1,他引:0
George S. Greaney George N. Somero 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1980,137(2):115-121
Summary The apparent Michaelis constant (K
m) of NADH for muscle-type (M4 isozyme) lactate dehydrogenases (LDHs) is highest, at any given temperature of measurement, for LDHs of cold-adapted vertebrates (Table 1). However, these interspecific differences in theK
m of NADH are not due to variations in LDH-NADH binding affinity. Rather, theK
m differences result entirely from interspecific variation in the substrate turnover constant (k
cat) (Fig. 1; Table 2). This follows from the fact that theK
m of NADH is equal tok
cat divided by the on constant for NADH binding to LDH,k
1, so that interspecific differences ink
cat, combined with identical values fork
1 among different LDH reactions, make the magnitude of theK
m of NADH a function of substrate turnover number. The temperature dependence of theK
m of NADH for a single LDH homologue is the net result of temperature dependence of bothk
cat andk
1 (Figs. 3 and 4). Temperature independentK
m values can result from simultaneous, and algebraically offsetting, increases ink
cat andk
1 with rising temperature. Salt-induced changes in theK
m of NADH also may be due to simultaneous perturbation of bothk
cat andk
1 (Table 3). These findings are discussed from the standpoint of the evolution of LDH kinetic properties, particularly the interspecific conservation of catalytic and regulatory functions, in differently-adapted species. 相似文献
18.
《Biochimica et Biophysica Acta (BBA)/General Subjects》2001,1525(1-2):29-36
The action of pancreatic α-amylase (EC 3.2.1.1) on various starches has been studied in order to achieve better understanding of how starch structural properties influence enzyme kinetic parameters. Such studies are important in seeking explanations for the wide differences reported in postprandial glycaemic and insulinaemic indices associated with different starchy foodstuffs. Using starches from a number of different sources, in both native and gelatinised forms, as substrates for porcine α-amylase, we showed by enzyme kinetic studies that adsorption of amylase to starch is of kinetic importance in the reaction mechanism, so that the relationship between reaction velocity and enzyme concentration [E0] is logarithmic and described by the Freundlich equation. Estimations of catalytic efficiencies were derived from measurements of kcat/Km performed with constant enzyme concentration so that comparisons between different starches were not complicated by the logarithmic relationship between E0 and reaction velocity. Such studies reveal that native starches from normal and waxy rice are slightly better substrates than those from wheat and potato. After gelatinisation at 100°C, kcat/Km values increased by 13-fold (waxy rice) to 239-fold (potato). Phosphate present in potato starch may aid the swelling process during heating of suspensions; this seems to produce a very favourable substrate for the enzyme. Investigation of pre-heat treatment effects on wheat starch shows that the relationship between treatment and kcat/Km is not a simple one. The value of kcat/Km rises to reach a maximum at a pre-treatment temperature of 75°C and then falls sharply if the treatment is conducted at higher temperatures. It is known that amylose is leached from starch granules during heating and dissolves. On cooling, the dissolved starch is likely to retrograde and become resistant to amylolysis. Thus the catalytic efficiency tends to fall. In addition, we find that the catalytic efficiency on the different starches varies inversely with their solubility and we interpret this finding on the assumption that the greater the solubility, the greater is the likelihood of retrogradation. We conclude that although α-amylase is present in high activity in digestive fluid, the enzymic hydrolysis of starch may be a limiting factor in carbohydrate digestion because of factors related to the physico-chemical properties of starchy foods. 相似文献
19.
Abstract Acetylcholinesterase (AChE) in the susceptible (S) and the resistant (R) strains of housefly (Musca domestica) was investigated using kinetic analysis. The Vmax values of AChE for hydrolyzing acetylthiocholine (ATCh) and butyrylthiocholine (BTCh) were 4578.50 and 1716.08nmol/min/mg* protein in the R strain, and were 1884.75 and 864.72 nmol/min/mg. protein in the Sstrain, respectively. The Vmax ratios of R to S enzyme were 2.43 for ATCh and 1.98 for BTCh. The Km values of AChE for ATCh and BTCh were 0.069 and 0.034 mmol/L in the S strain, and 0.156, 0.059 mmol/L in the R strain, respectively. The Km ratios of R to S enzyme were 2.26 for ATCh and 1.74 for BTCh. The ki ratios of S to R enzyme for three insecticides propoxur, methomyl and paraoxon were 46.04, 4.17 and 2. 86, respectively. In addition, kcat and kcat/Km for measuring turnover and catalytic efficiency of AChE were determined using eserine as titrant. The kcat values of AChE from the R strain for both ATCh and BTCh were higher than those values from the S strain. But the values of kcat/Km were in contrary to the kcat values with R enzyme compared to S enzyme. The AChE catalytic properties and sensitivity to the inhibition by three insecticides in the R and S strains of housefly were discussed based on contribution of Vmax, Km, ki, kcat and kcat/Km. All these data implied that AChE from the R strain might be qualitatively altered. We also observed an intriguing phenomenon that inhibitors could enhance the activity of AChE from the resistant strain. This “flight reaction” of the powerful enzyme might be correlated with the developing resistance of housefly to organophosphate or carbamate insecticides. 相似文献
20.
Takayoshi Tagami Keitaro Yamashita Masayuki Okuyama Haruhide Mori Min Yao Atsuo Kimura 《The Journal of biological chemistry》2015,290(3):1796-1803
The α-glucosidase from sugar beet (SBG) is an exo-type glycosidase. The enzyme has a pocket-shaped active site, but efficiently hydrolyzes longer maltooligosaccharides and soluble starch due to lower Km and higher kcat/Km for such substrates. To obtain structural insights into the mechanism governing its unique substrate specificity, a series of acarviosyl-maltooligosaccharides was employed for steady-state kinetic and structural analyses. The acarviosyl-maltooligosaccharides have a longer maltooligosaccharide moiety compared with the maltose moiety of acarbose, which is known to be the transition state analog of α-glycosidases. The clear correlation obtained between log Ki of the acarviosyl-maltooligosaccharides and log(Km/kcat) for hydrolysis of maltooligosaccharides suggests that the acarviosyl-maltooligosaccharides are transition state mimics. The crystal structure of the enzyme bound with acarviosyl-maltohexaose reveals that substrate binding at a distance from the active site is maintained largely by van der Waals interactions, with the four glucose residues at the reducing terminus of acarviosyl-maltohexaose retaining a left-handed single-helical conformation, as also observed in cycloamyloses and single helical V-amyloses. The kinetic behavior and structural features suggest that the subsite structure suitable for the stable conformation of amylose lowers the Km for long-chain substrates, which in turn is responsible for higher specificity of the longer substrates. 相似文献